Cloning restriction and modification genes

ABSTRACT

Methods for cloning restriction enzymes and their corresponding modification enzymes by selecting clones resistant to digestion by the restriction enzyme and subsequent selection of clones containing the restriction gene.

This is a continuation of copending application(s) Ser. No. 06/707,079filed on Mar. 1, 1985 now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to clones which produce restriction enzymes andmodification enzymes, to methods of producing such clones and to methodsof producing the restriction and modification enzymes from the clones.This invention also relates, more specifically, to clones for Hae II, M.Hae II, Taq I and M. Taq I and related methods for the production ofthese clones and enzymes.

Restriction endonucleases are a class of enzymes that occur naturally inbacteria. When they are purified away from other contaminating bacterialcomponents, restriction endonucleases can be used in the laboratory tobreak DNA molecules into precise fragments. This property enables DNAmolecules to be uniquely identified and to be fractionated into theirconstituent genes. Restriction endonucleases have proved to beindispensable tools in modern genetic research. They are the biochemical`scissors` by means of which genetic engineering and analysis isperformed.

Restriction endonucleases act by recognizing and binding to particularsequences of nucleotides (the `recognition sequence`) along the DNAmoleule. Once bound, they cleave the molecule within, or to one side of,the sequence. Different restriction endonucleases have affinity fordifferent recognition sequences. Close to one hundred differentrestriction endonucleases have been identified among the many hundredsof baterial species that have been examined to date.

Bacteria tend to possess at most only a small number of restrictionendonucleases per species. The endonucleases typically are namedaccording to the bacteria from which they are derived. Thus, the speciesHaemophilus aegyptius, for example, synthesizes 3 different restrictionendonucleases, named Hae I, Hae II and Hae III. Those enzymes recognizeand cleave the sequences (AT)GGCC(AT), PuGCGCPy and GGCC respectively.Escherichia coli RY13, on the other hand, synthesizes only one enzyme,EcoR I, which recognizes the sequence GAATTC.

In nature, restriction endonucleases play a protective role in thewelfare of the bacterial cell. They enable bacteria to resist infectionby foreign DNA molecules like viruses and plasmids that would otherwisedestroy or parasitize them. They achieve this resistance by scanning thelengths of the infecting DNA molecule and cleaving them each time therecognition sequence occurs. The break-up that takes place disables manyof the infecting genes and renders the DNA susceptible to furtherdegradation by non-specific exonucleases.

A second component of bacterial protective systems are the modificationgenes or methylases. These enzymes are complimentary to restrictionendonucleases and they provide the means by which bacteria are able toidentify their own DNA and distinguish it from foreign, infecting DNA.Modification methylases recognize and bind to the same nucleotiderecognition sequences as the corresponding restricting endonucleases,but instead of breaking the DNA, they chemically modify one or other ofthe nucleotides within the sequence by the addition of a methyl group.Following this methylation, the recognition sequence is no longer boundor cleaved by the restriction endonuclease. The DNA of a bacterial cellis always fully modified, by virtue of its modification methylases, thatit is therefore completely insensitive to the presence of the endogenousrestriction endonucleases. It is only unmodified, and thereforeidentifiably foreign DNA and is sensitive to restriction endonucleaserecognition and attack.

With the advent of genetic engineering technology, it is now possible toclone genes in order to produce proteins and enzymes encoded by the genein greater quantities than conventional purification techniques. The keyto cloning and isolating restriction clones is to develop a simple andreliable method to identify such clones within complex `libraries`, i.e.populations of clones derived by `shotgun` procedures when they occur atfrequencies as low as 10⁻⁴ to 10⁻³. Preferably, the method should beselective, such that the unwanted majority of clones are destroyed whilethe rare desirable clones survive.

Some investigators have used bacteriophage infection as a means ofselectively isolating restriction endonuclease clones (Walder et al.,Proc. Nat. Acad. Sci. 74 1503-1507 (1981), Mann et al., Gene 3: 97-112(1981). Since the presence of restriction-modification systems inbacteria enable them to resist infection by bacteriophages, cells thatcarry cloned restriction-modification genes can in principle beselectively isolated as survivors from libraries that have been exposedto phage. This method has been found, however, to have only limitedvalue. Specifically, it has been found that clonedrestriction-modification genes do not manifest sufficient phageresistance to confer selective survival.

Because purified restriction endonucleases, and to a lesser extentmodification methylases, are useful tools for characterizing andre-arranging DNA in the laboratory, there is a commercial incentive todevelop strains of bacteria that synthesize these enzymes in abundance.Such strains would be useful because they would simplify the task ofpurification as well as providing the means for production incommercially useful amounts.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided a novelapproach to the overproduction of restriction enzymes and theircorresponding modification enzymes by cloning genes that encode them andarranging for the genes to be expressed at elevated levels. Morespecifically, there is provided methods of cloning these enzymes, theclones so produced and methods of producing the enzymes themselves whichcomprises forming a library containing the DNA coding for the desiredrestriction enzyme, isolating those clones which contain thecorresponding modification gene, and screening clones containing themodification gene, for the presence of the restriction gene. Theapplication of this method to the Hae II and Taq I restriction andmodification genes of Haemophilus aegyptius and Thermus aquaticusrespectively is described in detail, together with the resulting strainsthat form the basis of a new and useful process for purifying the Hae IIand Taq I restriction and modification enzymes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the scheme for cloning Hae I restriction/modificationgenes.

FIG. 2 shows reproduction of photographs of gels of Hind III digests ofseveral Hae II restriction/modification and Hae III modification clones.

FIG. 3 illustrates the organization of Hae II and Hae IIIrestriction/modification genes of clones produced in accordance with thepresent invention.

FIG. 4 illustrates the organization of the plasmid pHa IIrestriction/modification gene fragments as well as a reproduction of aphotograph of a gel for Hind III double digests of pHae II.

FIG. 5 illustrates a first approach to the overexpression of Hae II.

FIG. 6 illustrates a second approach to the overexpression of Hae II.

FIG. 7 illustrates under- and over-producing plasmids for Hae IIrestriction and modification genes and the tabulation of the enzymeyields.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel approach for cloning restrictiongenes and harvesting restriction enzymes and their correspondingmodification enzymes from clones produced thereby. This approach takesadvantage of the fact that clones which contain modification genes willmethylate their own DNA, and in particular the corresponding restrictiongene's recognition sequence if it is present in the clone. Such cloneswill therefore be resistant to digestion in vitro by the correspondingrestriction endonuclease. It therefore follows that restrictionendonuclease digestion of these clones will result in the survival ofmethylase-encoding clones. Moreover, if the methylase encoding clonealso contains the corresponding restriction gene then such clones willprovide the means for overexpressing and harvesting the restrictionenzyme itself.

While not wishing to be bound by theory, it is believed that restrictionendonuclease genes occur proximate to corresponding modificationmethylase genes in the bacterial chromosome, and that the two genes aretherefore likely to be linked together during cloning experiments. Thus,it is believed that clones that acquire methylase genes are quite likelyto simultaneously acquire the corresponding endonuclease gene providedthat the fragment of DNA they receive during cloning is reasonablylarge.

In accordance with the present invention, it has been found thatrestriction genes and their corresponding modification genes arephysically close in the DNA of many bacteria. This being the case, inpracticing the present invention, selection for methylase-containingcells can be used as a simple and reliable method for selectivelyco-isolating methylase and endonuclease clones. In brief, selection ofmethylase-carrying clones from libraries which also contain DNAfragments coding for the corresponding restriction genes frequentlyresults in the isolation of clones that carry both methylase andrestriction endonuclease gene corresponding to the same DNA sequence.Methylase-selection is therefore an indirect way of selectingrestriction endonuclease clones.

The methods described herein by which restriction genes are preferablycloned and expressed include the following steps:

1. The DNA of the bacterial species to be cloned is purified.

2. The DNA is digested partially with a convenient restrictionendonuclease.

3. The resulting fragments are ligated into a cloning vector, such aspBR322, and the mixture is used to transform an appropriate host cellsuch as E. coli cells.

4. The DNA/cell mixture is plated on antibiotic media selective fortransformed cells. After incubation, the transformed cell colonies arescraped together into a single culture, the primary cell library.

5. The recombinant plasmids are purified in toto from the primary celllibrary to make a primary plasmid library.

6. The plasmid library is then digested to completion in vitro with therestriction enzyme whose corresponding methylase gene is sought.Exonuclease and/or phosphatase may also be added to the digestion toenhance the destruction of non-methylase clones.

7. The digested pool is transformed into E. coli and transformedcolonies are again obtained by plating on antibiotic plates. Some ofthese colonies--secondary cell individuals--may be picked and their DNAanalyzed for the presence of the modification and/or restriction genes.The remaining colonies may be scraped together to form a secondary celllibrary from which a secondary plasmid library may be subsequentlyprepared.

8. The secondary plasmid library may be redigested with restrictionendonuclease (with or without exonuclease or phosphatase) to repeat theselection, leading to the recovery of tertiary cell individuals,tertiary cell libraries and tertiary plasmid libraries.

9. Each round of restriction endonuclease digestion causes selectivedestruction of non-methylase clones, and results in an increase in therelative frequency of the desired methylase-carrying clones.

10. Surviving colonies among the secondary and tertiary population arepicked and analyzed for the presence of the methylase gene. If it isfound to be present, they are further analyzed for the simultaneouspresence of the restriction gene that is presumed to be linked to themethylase gene.

11. Methylase screening may be performed by four simple tests:

(a) The recombinant plasmid DNA molecule of the clone may be purifiedand exposed to the selecting restriction endonuclease to establish thatit is resistant to digestion. Provided that the plasmid vector carriesseveral sites for that endonuclease, resistance indicates modification,rather than mutational site loss.

(b) The recombinant plasmid may be digested with the enzyme initiallyused to fragment the donor bacterial DNA. The fragments present in theclone should be comprehensible, sufficiently large to encode a methylasegene (i.e., over 1 Kilobase pair) and, most important, common to avariety of independently-formed clones: the same fragment or fragmentsshould be present among all the clones.

(c) The total chromosomal DNA of the clone may be purified and exposedto the selective restriction endonuclease. If the clone carries themethylase gene, the bacterial chromosome should be fully methylated and,like the plasmid, should be found to be resistant to digestion.

(d) The cell extract from the clone may be prepared and assayed in vitrofor methylase activity. (Methylase protection and radioactivelabelling.) Methylase activity should be found.

12. Restriction endonuclease screening may be carried out in two ways:

(a) The cell extract from the clone may be prepared and assayed in vitrofor its ability to digest sensitive DNA. Restriction endonucleaseactivity should be found.

(b) The cells themselves may be tested in vitro for their ability toresist phage infection. Resistance to phage infection indicates thepresence of a restriction-modification system.

Although the above-outlined steps represent the preferred mode forpracticing the present invention, it will be apparent to those skilledin the art that the above-described approach can vary in accordance withtechniques known in the art.

Clones containing the restriction and modification genes of Ban I, HhaII, Hind III and Msp I have also been produced in accordance with thepresent invention. The source of DNA containing the above genes wasBacillus aneurinolyticus (Ban I) (Institute of Applied Microbiology, IAM1027, Sugisaki, H., Maekawa, Y., Kanazawa S. and Takanami. M. (1982)Nucleic Acids Res. 10, 5747-5752), Haenophilus haemolyliticus (Hha II)(ATTCC 10014), Haemophilus influenzae (Hind III) (ATCC 33928), andMoraxella species (Msp I) (ATCC 53043).

It should be noted that occasionally, the general approach above may, onoccasion, yield no methylase clones at all. However, it has beengenerally found that it does yield methylase clones and among these,about half tend to also carry the corresponding restriction gene. Atpresent, it is not clear whether the occasional failures result fromtechnical difficulties or from fundmental biological problems such asnon-linkage, failure to express, etc.

The following examples are given to additionally illustrate embodimentsof the present invention as it is presently preferred to practice. Itwill be understood that these examples are illustrative, and that theinvention is not to be considered as restricted thereto except asindicated in the appended claims.

EXAMPLE I Cloning of the Hae II Restriction-Modification Genes FIG. 1illustrates the Hae II Methylase cloning scheme in accordance with theabove-described method for cloning restriction genes. The Hae II cloneswere prepared as follows:

1. DNA purification: To prepare the DNA of Haemophilus aegyptius (ATCC1116), 5 gm of freshly-grown cell paste was resuspended in 20 ml of 25%sucrose, 50 mM Tris, pH 8.0. 10 ml of 0.25M EDTA, pH 8.0, was added. Thesuspension was left on ice for two hours. Thereafter, 24 ml of lysis mix(1% Triton X-100, 50 mM Tris, pH 8.0, 67 mM EDTA) plus 5 ml of 10% SDSwas added and mixed to allow the cells to lyse. 70 ml of freshly67-equilibrated phenol was then added and the solution was emulsified byshaking. 70 ml of chloroform was added and the solution was againemulsified by shaking. The mixture was then centrifuged at 10K rpm for30 minutes and the viscous upper layer containing DNA was transferred toa fresh bottle and re-extracted with phenol/chloroform twice more. Theupper DNA layer was transferred to dialysis tubing and dialyzed againstfour changes of 1X DNA buffer (10 mM Tris, 1 mM EDTA, pH 8.0) over 24hours.

The dialysed DNA solution was transferred to a beaker and 1/100th volumeof 10 mg/ml RNase was added to achieve a final concentration of 100ug/ml. The solution was incubated at 37° for 1 hour to digest the RNA.5M NaCl was then added to achieve 0.4M final concentration and 0.55volumes of isopropanol was then layered on top of the solution. The DNAwas spooled out of this mixture with a glass rod, then dissolved in 15ml of 1X DNA buffer and stored at 4° C.

2. Partial digestion: The purified DNA was titrated with Hind III toachieve partial digestion as follows: 2.0 ml of DNA at 100 ug/ml in 10mM Tris pH 7.5, 10 mM MgCl₂, 50 mM NaCl, 10 mM mercaptoethanol bufferwas dispensed into ten, 200 ul aliquots. To the first tube was added 40units of Hind III to achieve 2 units per ug of DNA. To the second tubewas added 20 units of Hind III (1 unit/ug), and so on, each succeedingtube receiving half of the previous amount of Hind III. The tubes wereincubated at 37° C. for one hour, then heat-treated at 72° C. for 15minutes and 10 ul from each analyzed by agarose gel electophoresis.Tubes exhibiting moderate, but incomplete, digestion were chosen as thesource of partial digest fragments for cloning. (These were the 0.13unit/ug and 0.06 unit/ug tubes. The two solutions were mixed togetherand used as described below.)

3. Ligation: The fragmented DNA was ligated to pBR322 as follows: 4.0 ugof Hind III partially digested H. aegyptius DNA (40 ul) was mixed with2.0 ug of Hind III-cleaved and dephosphorylated pBR322 (10 ul). 10 ul of10X ligation mix (500 mM Tris, pH 7.5, 100 mM MgCl₂, 100 mM DTT, 5 mMATP) was added plus 40 ul of sterile distilled water to bring the finalvolume to 100 ul. 5 ul of T4 DNA ligase was added and the mixtureallowed to incubate at 16° C. for 4 hours. Ten, 10 ul quantities wereused to transform E. coli strain RR1 as follows: Each 10 ul aliquot wasmixed with 100 ul of SSC/CaCl₂ (50 mM NaCl, 5 mM Na₃ Citrate, 67 mMCaCl₂) on ice and 200 ul of ice-cold competent E. coli RR1 cells wasadded. After a 2-minute heat shock at 43° C., the cells were dilutedinto 5 ml of Luria-broth (L-broth) and grown to saturation at 37° C.

4. Primary Cell Library: The transformed cell cultures were centrifuged,resuspended in 250 ul volumes and plated onto Luria-agar (L-agar) platescontaining 100 ug/ml ampicillin. After overnight incubation at 37° C.,the plates were each flooded with 2.5 ml of 10 mM Tris, pH 7.5, 10 mMMgCl₂ and the transformed colonies were scraped together and pooled toform the primary cell library. 5. Primary Plasmid Library: The primaryplasmid library was prepared as follows: 2.5 ml of the primary celllibrary was innoculated into 500 ml of L-broth containing 100 ug/mlampicillin. The culture was shaken overnight at 37° C. then centrifugedat 4K rpm for 5 minutes. The supernatant was discarded and the cellpellet was resuspended in 10 ml of 25% sucrose, 50 mM Tris, pH 8.0, atroom temperature. 5ml of 0.25M EDTA, pH 8.0, was added, followed by 3 mlof 10 mg/ml lysozyme in 0.25M Tris, pH 8.0. The solution was left on icefor 1 hour, then 12 ml of lytic mix (1% Triton X-100, 50 mM Tris, pH8.0, 67 mM EDTA) was forcefully pipetted in and the cell suspensiongently swirled to achieve lysis. After lysis, the mixture wastransferred to a 50 ml plastic centrifuge tube and spun at 17K rpm, 4°C. for 45 minutes. The supernatant was removed with a pipette. 20.0 gmof solid CsCl was weighed into a 50 ml plastic screw-cap tube and 22.0gm of supernatant was pipetted into the tube and mixed. 1.0 ml ofethidium bromide solution (5 mg/ml ethidium bromide in 10 mM Tris, pH8.0, 1 mM EDTA, 100 mM NaCl) was added to the mixture. The solution wastransferred to two 5/8 in.×3in. polyallomer centrifuge tubes and sealed.These were then spun in the Ti70 rotor for 42 hours at 50K rpm, 17° C.To collect the plasmids, the tops of the tubes were pierced with ascalpel and the lower of the two flourescent DNA bands collected bysyringe under ultraviolet light. The lower band from both tubes wascombined into a screw-top glass tube and the ethidium bromide wasremoved by extracting four times with an equal volume of ice-coldN-Butanol.

The extracted solution was transferred to dialysis tubing and dialyzedfor 24 hours against 4 changes of 1X DNA buffer. The dialyzed DNAsolution was then transferred to a pre-weighed 50 ml sterile centrifugetube and its volume measured. 5M NaCl was added to a final concentrationof 0.4M, then 2 volumes of isopropanol was added and mixed. The solutionwas stored overnight at -20° C. to precipitate the DNA. Afterprecipitation, the solution was spun at 15K rpm, 0° C. for 15 minutesand the supernatant discarded. The tube was left on the bench to air-dryfor 15 minutes, then 750 ul of sterile distilled water was added. Afterthe pellet had dissolved, 8 ul of 100X DNA buffer was added and thesolution was transferred to an Eppendorf tube and stored at -20° C. TheDNA concentrations of plasmids prepared in this way were found to beapproximately 100 to 200 ug/ml.

6. Digestion of Plasmid Pool: The primary plasmid pool was digested todestroy non-Hae II methylase clones as follows: The plasmid DNA wasdiluted to 50 ug/ml in 10 mM Tris pH 7.5, 10 mM MgCl₂, 10 mMmercaptoethanol, 50 mM NaCl. A total of 500 ul was prepared anddispensed into 5 tubes, 100 ul each. 75 units of Hae II was added to thefirst tube to achieve 15 units/ug DNA. 38 units of Hae II were added tothe second tube and so on, each tube receiving half of the previousamount. The tubes were incubated at 37° C. for 1 hour.

7. Transformation: A 10 ul sample from each tube was used to transformE. coli RR1 in the manner described previously. The cell/DNA mixtureswere plated onto L-agar plates containing 100 ug/ml ampicillinimmediately after the heat step, without intermediate dilution andgrowth. After overnight incubation at 37° C., the plates were examined.Digestion of the plasmid library with Hae II was found to have reducedthe number of transformants (i.e. the number of intact plasmids) by afactor of about 10². In later experiments, it was found that theaddition of 2.5 units of exonuclease III (New England Biolabs, Inc.,also available from BRL and IBI) or lambda exonuclease to each of thedigestion tubes described above (section 6) enhanced the destruction ofnon-methylase clones and reduced the number of transformants by a factorof about 10³ to 10⁴. Approximately 30 individual colonies were pickedfrom among the surviving colonies on the plates that had suffered thegreatest attrition (15 units Hae II/ug and 7.5 units Hae II/ug, plus orminus exonuclease). Each colony was inoculated into 10 ml of L-brothcontaining ampicillin to prepare a miniculture and was streaked ontoL-agar plates containing ampicillin to prepare a master stock.

8. Secondary Populations: The remaining colonies were scraped togetherto form a secondary cell library. This was used to prepare a secondaryplasmid library in the same manner a described for the preparation ofthe primary plasmid library.

9. Analysis of Secondary Plasmid Libraries: The secondary Hae IIlibraries were not used further in this particular experiment. As ageneral rule, however, it is helpful to analyze secondary plasmidlibraries by digestion and gel electrophoresis. Such analysis can beuseful for determining whether a significant proportion of thepopulation carries a common fragment and exhibits resistance torestriction endonuclease digestion.

10. Analysis of secondary individuals: Approximately 30 of the survivingcolonies among the secondary cell individuals were grown up into 10 mlcultures (section 7) and the plasmids that they carried were prepared bythe following miniprep purification procedure, adapted from the methodof Birnboin and Doly (Nucleic Acids Res. 7: 1513 (1979)).

Miniprep Procedure: Each culture was processed as follows: The 10 mlovernight culture was pelleted at 8K rpm for 5 minutes. The supernatantwas poured off and the cell pellet was resuspended in 1.0 ml of 25 mMTris, 10 mM EDTA, 50 mM glucose, pH 8.0, containing 1 mg/ml lysozyme.After 10 minutes at room temperature, 2.0 ml of 0.2M NaOH, 1% SDS wasadded and the tube was shaken to lyse the cells, then placed on ice.Once the solution had cleared, 1.5 ml of 3M sodium acetate, pH 4.8, wasadded and shaken. The precipitate that formed was spun down at 15K rpm,4° C. for 10 minutes. The supernatant was poured into a centrifuge tubecontaining 3 ml of isopropanol and mixed. After 10 minutes at roomtemperature, the tube was spun at 15K rpm for 10 minutes to pellet theprecipitated nucleic acids. The supernatant was discarded and the pelletwas air-dried at room temperature for 30 minutes. Once dry, the pelletwas resuspended in 850 ul of 10 mM Tris, 1 mM EDTA, pH 8.0. 75 ul of 5MNaCl was added and the solution was transferred to an Eppendorf tubecontaining 575 ul of isopropanol and again precipitated for 10 minutesat room temperature. The tube was then spun for 45 seconds in amicrofuge, the supernatant was discarded and the pellet was air-dried.The pellet was then dissolved in 50 ul of 10 mM Tris, 1 mM EDTA, pH 8.0,containing 100 ug/ml RNase and incubated for 1 hour at 37° C. to digestthe RNA. The DNA was precipitated once more by the addition of 50 ul of5M NaCl followed by 350 ul of isopropanol. After 10 minutes at roomtemperature, the DNA was spun down by centrifugation for 45 seconds, thesupernatant was discarded and the pellet was redissolved in a finalsolution of 150 ul of 10 mM EDTA, pH 8.0. The plasmid minipreps weresubsequently analyzed by digestion with Hind III and Hae II.

11. Methylase Gene Clones: Many of the plasmids that were analyzed werefound to carry random, Hind III fragments of Haemophilus DNA and to besensitive to digestion by Hae II. These plasmids were spurious survivorsof no further interest. (Their presence was found to be markedly reducedin later experiments in which exonuclease was used during the Hae IIdigestion-selection stage.) The remaining plasmids, however, were foundto be both resistant to Hae II and to carry at least two Hind IIIfragments of approximately 3.1 Kb and 2.9 Kb in length. These plasmidswere subsequently shown to carry both the Hae II modification andrestriction endonuclease genes.

In a parallel series of experiments, clones carrying the Hae IIImethylase genes were also selected and isolated (FIG. 1). These cloneswere found to carry a Hind III fragment of approximately 4.8 Kb. Severalclones were isolated that carried both the Hae III and the Hae IImethylase genes, and these were found to carry both the 4.8 Kb fragmentand the two 3.1 Kb and 2.9 Kb fragments. The isolation of these latterclones suggests that in H. aegyptius the Hae II restriction andmodification genes are linked to at least the Hae III methylase gene. Noclones were isolated which carried the Hae III restriction gene. FIG. 2is a reproduction of a photograph of a gel of Hind III-digests of someof the Hae II restriction/modification and the Hae III modificationclones. FIG. 3 summarizes the composition of some of the clones deducedfrom this, and similar analysis.

The clones that carried at least the 3.1 Kb and 2.9 Kb Hind IIIfragments (Hae II 4m-1, 4-8, 4-9, 4-10, 4-5, 4-11, 4-3 etc.) were judgedto carry the Hae II methylase gene on the basis of (a) insensitivity todigestion by the Hae II restriction endonuclease and (b) in vitro assaysof Hae II modification methylase activity. The methylase assays wereperformed as follows:

Methylase Assays: To assay for methylation three solutions wereprepared:

10X Methylation buffer: 0.5M Tris, pH 7.5, 100 mM EDTA, 50 mMMercaptoethanol.

Methylation reaction mix: Prepared fresh, for the analysis of 1 clone:100 ul lambda DNA (500 ug/ml), 100 ul 10X methylation buffer, 1 ul 100mM S-Adenosyl Methionine, 800 ul distilled water.

2X Hae II conversion buffer: 50 mM NaCl, 40 mM, MgCl₂, 15 mMMercaptoethanol.

Cell extracts were prepared as follows: A 100 ml culture of the clone tobe tested was grown overnight in L-broth plus 100 ug/ml ampicillin at37° C. and the cells pelleted by centrifugation at 4K rpm for 5 minutes.The supernatant was discarded and the pellet was resuspended in 5 ml ofsonication buffer (10 mM Tris, pH 7.5, 10 mM Mercaptoethanol, 1 mMEDTA). Once resuspended, 0.5 ml of sonication buffer containing 10 mg/mllysozyme was added. The suspension was swirled and left on ice for 1hour. A 1 ml sample was transferred to an Eppendorf tube and sonicatedgently for two 10-second bursts to disrupt the cells. The tube wa spunfor 30 seconds in a microfuge and the supernatant was used as the cellextract.

To assay the extract, the methylation reaction mix was dispensed into 5tubes, 150 ul into the first tube, and 102.5 ul into each of theremaining 4 tubes. 7.5 ul of the cell extract was added to the firsttube, mixed, and 47.5 ul was removed and added to the next tube, mixedand so on. The first tube thus received 1 ul of extract per ug of DNA,the second tube, 0.3 ul/ug, the third tube, 0.1 ul/ug and so on. Thetubes, each now containing 100 ul, were incubated at 37° C. for onehour, then heated to 72° C. for 10 minutes to stop the reactions. 100 ulof 2X conversion buffer, and 25 units of Hae II restriction enzyme, werethen added to each tube. The solutions were again incubated at 37° C.for one hour, then a 20 ul sample of each was analyzed by gelelectrophoresis. The clones were found to synthesize about 5000 units ofHae II methylase per gram of wet cell paste.

12. Restriction of Gene Clones: The clones identified above (section 11)as carrying the Hae II modification methylase gene were also found tocarry the Hae II restriction endonuclease gene. This was established byin vitro restriction endonuclease assays performed as follows:

Endonuclease Assays: To assay for endonuclease activity, two solutionswere prepared:

10X Hae II Restriction endonuclease buffer: 100 mM Tris, pH 7.5, 100 mMMgCl₂, 100 mM Mercaptoethanol, 500 mM NaCl. Digestion reaction mix:Prepared fresh, for the analysis of 1 clone: 100 ul lambda DNA (500ug/ml), 100 ul 10X Hae II Restriction endonuclease buffer 800 uldistilled water.

The cell extract was prepared in the manner described above for themethylation assay (section 11). To assay the extract, the digestionreaction mix was dispensed into 6 tubes, 150 ul into the first tube and102.5 ul into each of the remaining 5 tubes. 7.5 ul of the extract wasadded to the first tube, mixed and so on. The first tube thus received 1ul of extract per ug of DNA, the second tube, 0.3 ul/ug, the third tube,0.1 ul/ug and so on. The tubes, each now containing 100 ul, wereincubated at 37o for one hour, then a 20 ul sample of each was analyzedby gel electrophoresis. The clones were found to synthesize about 3000units of Hae II restriction endonuclease per gram of wet cell paste.

In tests with phages, the clones were found to resist phage infection toonly a slight degree: The efficiency of plating of phage lambda wasfound to be between 0.1 and 0.5. (Clone 4m-1 (FIGS. 2 and 3) wasexceptional. The efficiency of plating of phage lambda on 4m-1 was lessthan 10⁻⁴.).

EXAMPLE II Overexpression of the Hae II Restriction and ModificationGenes

1. One of the clones obtained in Example I, designated pHae II 4-11(FIGS. 3 and 4), was used for further analysis and for overexpressionbecause it possessed the simplest structure. FIG. 4 shows a simplifiedrestriction map of the two inserted Hind III fragments in this clone.The map was established by conventional double digest procedures.

2. Two different procedures were devised to achieve overproduction(FIGS. 5 and 6). Both procedures involved joining the Hind III fragmentsto a regulatory element that included a powerful promotor such that whenthe promoter was derepressed, transcription of the restriction andmodification genes would take place at an exceptionally high rate. Suchoverexpression systems have been previously described.

3. In the first procedure, the 4-11 plasmid was cleaved with Hae III toexcise the Hind III fragments together with a little terminal pBR322DNA, in one segment. The vector pGW7 (ATCC 40166, also available fromNew England Biolabs) was digested with BamH I and the cohesive BamH Itermini filled in with DNA polymerase. For the Hae II plasmid, 12.5 ugof plasmid DNA was mixed with 80 units of Hae III restrictionendonuclease in 10 mM Tris, pH 7.5, 10 mM MgCl₂, 10 mM mercaptoethanol,50 mM NaCl, to a final volume of 500 ul. For the pGW7 plasmid, 12.5 ugof plasmid DNA was mixed with 80 units of BamH I restrictionendonuclease in the same buffer, but to a volume of 250 ul. The twodigestions were incubated at 37° C. for one hour then terminated byheating at 72° C. for 10 minutes. The BamH I digestion was subsequentlyfilled in as follows: To 100 ul of the digestion was added 30 ul of 5XdNTP stock solution (25 mM dCTP, 25 mM dGTP, 25 mM dTTP), 5 ul of 10XPolymerase buffer (100 mM Tris, pH 7.5, 100 mM MgCl₂, 10 mMDTT, 500 mMNaCl), 7.5 ul of distilled water, and 7.5 ul of DNA Polymerase Klenowfragment. The reaction was incubated at 20° C. for 15 minutes. 45 ulwere then withdrawn and mixed with 45 ul of the 4-11 plasmid digestion,10 ul of 10X ligation mix (described in Section 3) and 5 ul of T4 DNAligase. The ligation reaction was incubated for 3 hours at 20° C. 10 ulquantities of the ligation were then transformed into E. coli RR1 andplated onto L-agar plates containing amplicillin.

The plates were incubated overnight at 30° C.: The whole ligationyielded 8 plates with approximately 250 colonies/plate, i.e., about 2000recombinants in all. The plates were scraped and a library of plasmidsprepared in the manner described for the preparation of primary plasmidlibraries (sections 4 and 5 of Example I). The library was digested withHae II restriction endonuclease to selectively destroy recombinantplasmids that did not carry the Hae II modification gene. (2.5 units ofExonuclease III was added to each of the digestions to reduce thebackground of spurious survivors). Following transformation into E. coliRR1 , plating on L-agar plates containing ampicillin and incubation at30° C., surviving colonies were picked and the plasmids that theycarried were purified by the miniprep procedure (section 10 of ExampleI) and then analyzed by digestion and gel electrophoresis. FIG. 5depicts the entire experimental scheme and also shows a gel ofdigestions of some of the survivors.

One of the clones isolated by this procedure, designated pHaeII 7-11 wasfound to carry the Hae II genes in one orientation, B, with respect tothe P_(L) promoter. Several other clones, typical of which is pHaeII7-6X carried the genes in the other, A, orientation. The clones 7-11 and7-6X were assayed to determine which, if either, overproduced theendonuclease and the methylase when the external, P_(L), promoter wasdepressed by temperature induction. The induction experiments wereperformed in the following way:

10 ml cultures of the clones were grown overnight at 30° C. in L-brothcontaining 100 ug/ml ampicillin. The following morning each culture wasdiluted 50-fold into 500 ml of fresh L-broth and grown in a shakingincubator at 30° C. After approximately 6 hours of incubation 200 ml ofeach culture was withdrawn, and its optical density at 590 nm (OD590)was measured. The withdrawn cells were collected by centrifugation for15 minutes at 10K rpm. The rest of each culture was then shifted to atemperature of 43° C. to derepress the P_(L) promoter. After 3 hours ofvigorous shaking at this temperature, the OD₅₉₀ of each culture wasagain measured and 200ml of each culture was again collected bycentrifugation.

The cell pellets were stored at -20° C. until it was convenient to assaythem.

To assay for methylase and endonuclease activities, the pellets werethawed and resuspended in sufficient sonication buffer containing 1mg/ml lysozyme to reach an estimated OD₅₉₀ of 50. The cell suspensionswere left on ice for 1 hour then sonicated and assayed as describedabove (sections 11 and 12 of Example 1).

Clone pHaeII 7-11 was found to overproduce both the Hae II restrictionendonclease and the Hae II modification methylase when the culture wasshifted to high temperature. Up to 10⁶ units of endonuclease, and 3×10⁴units of methylase, per gram of wet cell paste, was produced by the 7-11clone. Conversely, pHaeII 7-6X was found to underproduce both enzymeswhen the P_(L) promotor was derepressed. The underproduction isconsistent with the reversed orientation that the two genes bear toP_(L) in this plasmid.

pHae II 7-11 is a new and useful clone from which the Hae II restrictionendonuclease and modification methylase enzymes can be purified inquantity.

4. A second experimental procedure to join the Hae II genes to theoverexpression promotor, P_(L) was devised and carried out. Theprocedure is simpler and more reliable than the procedure described inthe last section, but it yields clones in only one orientation. As itturned out, the orientation it yielded, B, was the one desired. Theprocedure is outlined in FIG. 6. It used the pGW10 expression vector(ATCC 40167, also available from New England Biolabs), and the desiredclones were isolated by direct selection on L-agar plates containingtetracycline. The procedure generated several overproducing plasmids,one of which is pHae II 10-3. After temperature induction, clone 10-3behaves like 7-11 and overproduces both the methylase and theendonuclease to similar levels as clone 7-11.

FIG. 7 summarizes the structures of the under- and overproducingplasmids described above and tabulates the enzyme yields.

EXAMPLE III Cloning of the Taq I Restriction-Modification Genes

1. DNA purification: To prepare the DNA of Thermus Aquaticus YT1 (ATCC25104), 5 gm of freshly-grown cell paste was resuspended in 20 ml of 25%sucrose, 50 mM Tris, pH 8.0. 10 ml of 0.25M EDTA, pH 8.0, plus 6 ml of10 mg/ml lysozyme in 0.25M Tris, pH 8.0, was added. The suspension wasleft on ice for two hours, then 24 ml of lysis mix (1% Triton X-100, 50mM Tris, pH 8.0, 67 mM EDTA) plus 5 ml of 10% SDS was added and mixed toinduce cell lysis. 70 ml of freshly-equilibrated phenol was then addedand the solution was emulsified by shaking. 70 ml of chloroform wasadded and the solution was again emulsified by shaking. The mixture wasthen centrifuged at 10K rpm for 30 minutes and the viscous upper layercontaining DNA was transferred to a fresh bottle and re-extracted withphenol/chloroform twice more. The upper DNA layer was transferred todialysis tubing and dialyzed against four changes of 1X DNA buffer (10mM Tris, 1 mM EDTA, pH 8.0) over 24 hours.

The dialyzed DNA solution was transferred to a beaker and 1/100th volumeof 10 mg/ml RNase was added to achieve a final concentration of 100ug/ml. The solution was incubated at 37° C. for 1 hour to digest theRNA. 5M NaCl was then added to achieve 0.4M final concentration and 0.55volumes of isopropanol was then layered on top of the solution. The DNAwas spooled out of this mixture with a glass rod, then dissolved in 15ml of 1X DNA buffer and sorted at 4° C.

2. Partial digestion: The purified DNA was titrated with BamH I toachieve partial digestion as follows: 2.0 ml of DNA at 100 ug/ml in 10mM Tris pH 7.5, 10 mM MgCl₂, 10 mM mercaptoethanol, 50 mM NaCl bufferwas dispensed into ten, 200 ul aliquots. To the first tube was added 200units of BamH I to achieve 10 units/ug of DNA. To the second tube wasadded 100 units of BamH I (5 units/ug), and so on, each succeeding tubereceiving one half of the previous amount of BamH I. The tubes wereincubated at 37° C. for one hour, then heated to 75° C. for 15 minutesto terminate the reactions. 10 ul from each tube was then analyzed byagarose gel electrophoresis. Tubes exhibiting moderate, but incomplete,digestion were chosen as the source of partially-digested fragments forcloning. (These were the 1.25, 0.63, 0.3 and 0.15 unit/ug tubes. Thefour solutions were mixed together and used as described below).

3. Ligation: The fragmented DNA was ligated to pBR322 as follows: 6.7 ugof BamH I partially-digested T. aquaticus DNA (67 ul) was mixed with 2.5ug of BamH I-cleaved and dephosphorylated pBR322 (25 ul). 25 ul of 10Xligation mix (500 mM Tris, pH 7.5, 100 mM MgCl₂, 100 mM DTT, 5 mM ATP)was added plus 133 ul of sterile distilled water to bring the finalvolume to 250 ul. 5 ul of T4 DNA ligase at 4×10⁵ units per ml was addedthen the mixture was incubated at 17° C. for 4 hours. 20 ul ofchloroform was then added and the solution was briefly shaken andcentrifuged to terminate the reaction and to sterlize the solution. 100ul of the solution was mixed with 1.0 ml of SSC/CaCl₂ (50 mM NaCl, 5 mMNa₃ Citrate, 67 mM CaCl₂) on ice and 2.0 ml of ice-cold competent E.coli RR1 cells were added. The mixture was heated to 43° C. for 5minutes then diluted by the addition of 10 ml of Luria-broth (L-broth)and incubated at 37° C. for 4 hours.

4. Primary Cell Library: The transformed culture was centrifuged brieflyand the supernatant was discarded. The cell pellet was then resuspendedin 2.0 ml of L-broth and 200 ul quantities were plated onto each of 10Luria-agar (L-agar) plates containing 100 ug/ml ampicillin. Afterovernight incubation at 37° C., the plates were each flooded with 2.5 mlof 10 mM Tris, pH 7.5, 10 mM MgCl₂ and the transformant colonies werescraped together and pooled to form the primary cell library.

5. Primary Plasmid Library: The primary plasmid library was prepared asfollows: 2.5 ml of the primary cell library was innoculated into 500 mlof L-broth containing 100 ug/ml ampicillin. The culture was shakenovernight at 37° C. then centrifuged at 4K rpm for 5 minutes. Thesupernatant was discarded and the cell pellet was resuspended in 10 mlof 25% sucrose, 50 mM Tris, pH 8.0, at room temperature. 5 ml of 0.25MEDTA, pH 8.0, was added, followed by 3 ml of 10 mg/ml lysozyme in 0.25MTris, pH 8.0. The solution was left on ice for 1 hour then 12 ml oflytic mix (1% Triton X-100, 50 mM Tris, pH 8.0, 67 mM EDTA) wasforcefully pipetted in and the cell suspension was gently swirled toachieve lysis. The lysed mixture was transferred to a 50 ml plasticcentrifuge tube and spun at 17 K rpm, 4° C. for 45 minutes. 22.0 gm ofthe supernatant was removed by pipet and transferred to a 50 ml plasticscrew-cap tube. 20.0 gm of solid CsCl, and 1.0 ml of 5 mg/ml ethidiumbromide in 10 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl were added. Thetube was gently shaken until all the CsCl had dissolved, then thesolution was transferred to two 5/8 in.×3 in. polyallomerultracentrifuge tubes. The tubes were sealed then spun in the Ti70 rotorfor 42 hours at 50K rpm and 17° C. To collect the plasmids, the tops ofthe tubes were pierced with a scalpel and the lower of the twoflourescent DNA bands collected by syringe under ultraviolet light. Thelower band from both tubes was combined and the ethidium bromide wasremoved by extracting four times with an equal volume of ice-coldN-Butanol.

The extracted solution was transferred to dialysis tubing and dialyzedfor 24 hours against 4 changes of 1X DNA buffer (section 1). Thedialyzed DNA solution was then transferred to a pre-weighed 50 mlcentrifuge tube and its volume measured. 5M NaCl was added to a finalconcentration of 0.4M, then 2 volumes of isopropanol was added andmixed. The solution was stored overnight at -20° C. to precipitate theDNA. After precipitation, the solution was spun at 15K rpm, 0° C. for 15minutes and the supernatant was discarded. The tube was left to air-dryfor 15 minutes then 750 ul of sterile distilled water was added. Afterthe pellet had dissolved, 8 ul of 100X DNA buffer was added and thesolution was transferred to an Eppendorf tube and stored at -20° C. Theplasmid DNA concentraction was found to be 150 ug/ml.

6. Digestion of Plasmid Pool: The primary plasmid pool was digested todestroy non-Taq I methylase clones as follows: 150 ul of the plasmid DNAsolution was diluted to a volume of 450 ul by the addition of 45 ul of10X Taq I buffer (100 mM Tris, pH 8.4, 60 mM MgCl₂, 60 mMMercaptoethanol, 1M NaCl) and 255 ul of distilled water. The solutionwas then dispensed into 5 tubes: the first four tubes received 100 uleach and the final tube received 50 ul. 40 units of Taq I was added tothe first tube to achieve 8 units of enzyme per ug of DNA. 20 units ofTaq I were added to the second tube (4 units/ug) and so on, each tubereceiving half of the previous amount. The final tube served as anexperimental control and received no Taq I enzyme. The solutions wereoverlayed with 50 ul of parraffin oil to inhibit evaporation then thetubes were incubated at 65° C. for 1 hour.

7. Transformation: A 10 ul sample from each tube was used to transformE. coli RR1 in a manner similar to that described previously: each 10 ulsolution was mixed with 100 ul of SSC/CaCl₂ (50 mM NaCl, 5 mM Na₃Citrate, 67 mM CaCl₂) on ice and 200 ul of ice-cold competent E. coliRR1 cells were added. After a 3-minute heat shock at 43° C., thecell/DNA mixtures were immediately plated onto L-agar plates containing100 ug/ml ampicillin. The plates were incubated overnight at 37° C. thenthey were examined. Digestion of the plasmid library with Taq I wasfound to have reduced the number of transformants (i.e. the number ofintact plasmids) by a factor of between about 10³ and 10⁴. 28 individualcolonies from among the survivors on the plates were picked and each wasinoculated into 10 ml of L-broth containing ampicillin and streaked ontoL-agar plates containing ampicillin.

8. Secondary Population: The remaining colonies were scraped together toform a secondary cell library which was used to prepare a secondaryplasmid library in the manner described for the preparation of theprimary plasmid library.

9. Analysis of Secondary Plasmid Library: The secondary plasmid librarywas digested with BamH I and analyzed by gel electrophoresis. A single,prominant 5.5 Kilobase-pair fragment was found to be present within thepopulation.

10. Analysis of secondary individuals: 28 of the surviving coloniesamong the secondary cell individuals were grown up into 10 ml cultures(section 7) and the plasmids that they carried were prepared by theminiprep purification procedure described in Example I, section 10,above. The plasmid minipreps were analyzed by digestion with Taq I andBamH I.

11. Methylase Gene Clones: Some of the plasmids that were analyzed werefound to carry random, BamH I fragments of T. aquaticus DNA and to besensitive to digestion by Taq I. These plasmids were spurious survivorsand were of no further interest. The remaining plasmids, however, werefound to be resistant to digestion by Taq I and to carry at least onecommon BamH I fragment of approximately 5.5 Kb in length. All of theseplasmids were subsequently shown to carry both the Taq I modificationand restriction endonuclease genes. One of these plasmids designatedpTaq I 18, is an example of the simplest type of clone: it was found tocarry only the one, 5.5 Kb, BamH I fragment. Cells harboring pTaq I 18were found to synthesize both the Taq I restriction endonuclease andmodification methylase in abundance.

The assays to detect Taq I modification methylase activity in vitro wereperformed as follows:

Methylase Assays: To assay for methylation, three solutions wereprepared:

10X Methylation buffer: 0.5M Tris, pH 8.0, 100 mM EDTA, 50 mMMercaptoethanol.

Methylation reaction mix: 100 ul lambda DNA at 500 ug/ml, 100 ul 10Xmethylation buffer, 1 ul 100 mM S-Adenosyl Methionine, 800 ul distilledwater.

10X Conversion buffer: 0.5M NaCl, 0.3M MgCl₂, 50 mM Mercaptoethanol.

Cell extracts: A 100 ml culture was grown overnight at 37° C. in L-brothplus 100 ug/ml ampicillin. The cells were harvested the followingmorning by centrifugation at 4K rpm for 5 minutes. The supernatant wasdiscarded and the pellet was resuspended in 3.6 ml of sonication buffer(10 mM Tris, pH 7.5, 10 mM Mercaptoethanol, 1 mM EDTA). 0.4 ml ofsonication buffer containing 10 mg/ml lysozyme was added and thesuspension was swirled and left on ice for 1 hour. A 1 ml sample wasthen transferred to an Eppendorf tube and sonicated gently for two, 10second bursts to disrupt the cells. The tube was spun for 30 seconds ina microfuge to pellet the cell debris. The supernatant was transferredto a fresh Eppendorf tube and heated to 65° C. for 20 minutes and theprecipitate again removed by micro-centrifugation for 30 seconds. Thesupernatant that remained wa used as the cell extract.

Assays: To assay the extract, the methylation reaction mix was preparedfresh and dispensed into 5 tubes, 150 ul into the first tube, and 102.5ul into each of the remaining 4 tubes. 7.5 ul of the cell extract wasadded to the first tube, mixed and so on. The first tube thus received 1ul of extract per ug of DNA, the second tube, 0.3 ul/ug, the third tube,0.1 ul/ug and so on, and each tube finally contained about 100 ul ofsolution. 50 ul of paraffin oil was layered on top of each solution toinhibit evaporation and the tubes were incubated at 65° C. for one hour.11 ul of 10X Conversion buffer, and 25 units of Taq I restrictionenzyme, were then added to each tube. The solutions were again incubatedat 65° C. for one hour then a 20 ul sample of each was analyzed by gelelectrophoresis. The clones were found to synthesize about 2×10⁵ unitsof Taq I methylase per gram of wet cell paste.

12. Restriction Gene Clones: The clones that carried the 5.5 Kb BamH Ifragment were found to synthesize the Taq I restriction endonuclease aswell as the modification methylase.

The assays to detect Taq I restriction endonuclease activity in vitrowere performed as follows:

Endonuclease Assays: To assay for endonuclease activity, two solutionswere prepared:

10X Taq I endonuclease buffer: 100 mM Tris, pH 8.4, 60 mM MgCl₂, 60 mMMercaptoethanol, 1.0M NaCl.

Endonuclease reaction mix: 100 ul lambda DNA at 500 ug/ml, 100 ul 10XTaq I endonuclease buffer, 800 ul distilled water.

Cell extracts: Extracts were prepared in the manner described above forthe methylase assays (section 11).

Assays: To assay the extract, the endonuclease reaction mix was preparedfresh and dispensed into 5 tubes, 150 ul into the first tube, and 102.5ul into each of the remaining 4 tubes. 7.5 ul of the cell extract wasadded to the first tube, mixed, and 47.5 ul was removed and added to thenext tube, mixed and so on. The first tube thus received 1 ul of extractper ug of DNA, the second tube, 0.3 ul/ug, the third tube, 0.1 ul/ug andso on, and each tube finally contained about 100 ul of solution. 50 ulof paraffin oil was layered on top of each solution to inhibitevaporation and the tubes were incubated at 65° C. for one hour. A 20 ulsample of each was analyzed by gel electrophoresis. The clones werefound to synthesize about 2×10⁵ units of Taq I restriction endonucleaseper gram of wet cell paste. In tests with phages, the clones were notfound to resist phage infection to any measurable degree: The efficiencyof plating of phage lambda was found to be between 0.5 and 1.

What is claimed is:
 1. A method of isolating DNA coding for arestriction endonuclease comprising the steps of:(a) forming a DNAlibrary from a source containing DNA coding for the restrictionendonuclease; (b) digesting the DNA library of step (a) with anappropriate restriction enzyme to obtain DNA coding for thecorresponding methylase; (c) transforming a host cell with the DNA ofstep (b); (d) analyzing varying amounts of extract from the transformedhost cell of step (c) for restriction endonuclease activity; and (e)isolating recombinant DNA from the host cell of step (d) which exhibitsrestriction endonuclease activity.
 2. The method of claim 1, wherein theDNA library of step (a) is formed by the steps of:(a) purifying DNA fromthe source containing DNA coding for the restriction endonuclease; (b)digesting the purified DNA to form DNA fragments; (c) ligating the DNAfragments of step (b) into a cloning vector to form a recombinantvector; (d) transforming a host cell with the recombinant vector of step(c) to form a primary cell library; and (e) purifying recombinantvectors from the primary cell library to form a primary vector library.3. The method of claim 2, wherein the source containing the DNA codingfor the restriction endonuclease is a bacterium which produces therestriction endonuclease.
 4. The method of claim 2, wherein the cloningvector is a plasmid or viral DNA molecule.
 5. The method of claim 2,wherein the DNA which codes for the corresponding methylase is obtainedby digesting the primary vector library of step (e) to completion with arestriction endonuclease corresponding to the methylase encoded by theisolated DNA.
 6. The method of claim 5, where the digestion issupplemented by the addition of an exonuclease or phosphatase.
 7. Amethod of producing a restriction endonuclease comprising the stepsof:(a) purifying DNA from a source containing DNA coding for therestriction endonuclease; (b) digesting the purified DNA to form DNAfragments; (c) ligating the DNA fragments of step (b) into a cloningvector to form a recombinant vector; (d) transforming a host cell withthe recombinant vector of step (c) to form a DNA library; (e) digestingthe DNA library of step (d) with an appropriate restriction enzyme toobtain DNA coding for the corresponding methylase; (f) transforming ahost cell with the DNA of step (e); (g) analyzing varying amounts ofextract from the transformed host cell of step (f) for restrictionendonuclease activity; (h) culturing the host cell of step (g) whichexhibits restriction endonuclease activity; and (i) recovering therestriction endonuclease from the culture.
 8. The method of claim 7,wherein the source containing the DNA coding for the restrictionendonuclease is a bacterium which produces the restriction endonuclease.9. The method of claim 30, wherein the cloning vector is a plasmid orviral DNA molecule.
 10. The method of claim 7, wherein the DNA whichcodes for the corresponding methylase is obtained by digesting the DNAlibrary of step (e) to completion with a restriction endonucleasecorresponding to the methylase encoded by the DNA.
 11. The method ofclaim 7, where the digestion is supplemented by the addition of anexonuclease or phosphatase.